U.S. patent number 6,034,065 [Application Number 07/985,831] was granted by the patent office on 2000-03-07 for elucidation and synthesis of antineoplastic tetrapeptide phenethylamides of dolastatin 10.
This patent grant is currently assigned to Arizona Board of Regents. Invention is credited to Jozsef Barkoczy, George R. Pettit.
United States Patent |
6,034,065 |
Pettit , et al. |
March 7, 2000 |
**Please see images for:
( Certificate of Correction ) ** |
Elucidation and synthesis of antineoplastic tetrapeptide
phenethylamides of dolastatin 10
Abstract
Dolastatin 10, a linear pentapeptide, has shown potent
antineoplastic activity profiles against various experimental
cancer systems. The design and synthesis of structural
modifications of dolastatin 10 having significant antineoplastic
activity against human cancer cell lines has been accomplished.
Members of this group have demonstrated significant antineoplastic
activity against selected human cancer cell lines. Especially:
Ovarian OVSCAR-3; Central Nervous System ("CNS") SF295; Renal A498;
Lung NCI460; Colon KM20L2 and Melanoma SK-MEL-3.
Inventors: |
Pettit; George R. (Paradise
Valley, AZ), Barkoczy; Jozsef (Budapest, HU) |
Assignee: |
Arizona Board of Regents
(Tempe, AZ)
|
Family
ID: |
25531838 |
Appl.
No.: |
07/985,831 |
Filed: |
December 3, 1992 |
Current U.S.
Class: |
514/19.3;
530/330; 514/15.4; 514/19.2; 514/17.7; 514/18.6; 514/19.6 |
Current CPC
Class: |
C07K
5/0205 (20130101); A61P 35/00 (20180101); A61K
38/00 (20130101) |
Current International
Class: |
C07K
5/02 (20060101); C07K 5/00 (20060101); A61K
38/00 (20060101); A61K 038/00 () |
Field of
Search: |
;530/330 ;514/18 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
4978744 |
December 1990 |
Pettit et al. |
|
Other References
Correia Pharmac. Ther. vol. 52 (1991) pp. 127-147. .
Jacobsen et al. J. of the Natl. Cancer Institute vol. 83 (22) pp.
1672-1677 ..
|
Primary Examiner: Huff; Sheela
Attorney, Agent or Firm: Mybeck; Richard R. Scull; Peter
B.
Government Interests
This invention relates generally to the field of antineoplastic
compounds, and more particularly to the design and synthesis of
selected structural modifications of peptides isolated from the
Indian Ocean sea hare Dolabella auricularia, namely antineoplastic
tetrapeptide phenethylamides of dolastatin 10, which have been
found to demonstrate effective antineoplastic activity against
various human cancer cell lines. Financial assistance for this
project was provided by U.S. Government Grant Number
OIG-CA44344-01A1-2: the United States Government may own certain
rights to this invention.
Claims
We claim:
1. A composition of matter comprising a compound having the general
structure: ##STR4## wherein n is selected from the group consisting
of 1, 2, and 3.
2. A method of inhibiting the growth of abnormal human cells
selected from the group consisting of leukemia, non-small cell lung
cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, and
renal cancer; comprising engaging said cells with a growth
inhibitory amount of a compound having the following structural
formula: ##STR5## wherein n is selected from the group consisting
of 1, 2, and 3.
3. A method according to claim 2 wherein said abnormal cells are
selected from the group of abnormal cell lines having the following
NCI cell line designations: P-388, OVCAR-3, SF-295, A498, NCI 460,
KM20L2, and SK-MEL-3.
4. A method according to claim 3 wherein n=1.
5. A method according to claim 3 wherein n=3.
6. A method according to claim 2 wherein n=2.
7. A method according to claim 6 wherein said abnormal human cells
are selected from the group consisting of leukemia cell lines
having the NCI Cell Line designations: CCRF-CEM, HL-60(TB), K-562,
MOLT-4, RPM-8226 and SR.
8. A method according to claim 6 wherein said abnormal human cells
are selected from the group consisting of Non-Small Cell Lung
Cancer Cell Lines having the NCI Cell Line designations: A549/ATCC,
EXVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H460, NCI-H522, and
LXFL-529.
9. A method according to claim 6 wherein said abnormal cells are
selected from the group consisting of Colon Cancer Cell Lines
having the NCI Cell Line designations: C0L0 205, DLD-1, HCT-116,
HCT-15, KM20L2 and SW-620.
10. A method according to claim 6 wherein said abnormal cells are
selected from the group consisting of CNS Cancer Cell Lines having
the NCI Cell Line designations: SF-268, SF-539, SNB-19, SNB-75,
SNB-78 and U251.
11. A method according to claim 6 wherein said abnormal cells are
selected from the group consisting of Melanoma Cancer Cell Lines
having the NCI Cell Line designations: LOX1MV1, MALMB-3M, M14,
M19-MEL, SK-MEL-2, SK-MEL-5, and UACC-62.
12. A method according to claim 6 wherein said abnormal cells are
selected from the group consisting of Ovarian Cancer Cell Lines
having the NCI Cell Line designations: 1GROV1, OVCAR-5, OVCAR-8,
and SK-OV-3.
13. A method according to claim 6 wherein said abnormal cells are
selected from the group consisting of Renal Cancer Cell Lines
having the NCI Cell Line designations: 786-O, ACHN, CAK1-1,
RXP-393, SN12C, TK-10, and UO-31.
14. A method according to claim 3 wherein n=2.
15. A composition of matter according to claim 1 wherein n=1.
16. A composition of matter according to claim 1 wherein n=2.
17. A composition of matter according to claim 1 wherein n=3.
18. A pharmaceutical preparation comprising a compound having the
structure set forth in claim 1.
Description
BACKGROUND OF THE INVENTION
A great number of ancient marine invertebrate species in the Phyla
Bryozoa, Mollusca and Porifera were well established in the earth's
oceans over one billion years ago. Certainly such organisms had
explored trillions of biosynthetic reactions in their evolutionary
chemistry to reach present levels of cellular organization,
regulation and defense. Marine sponges have changed minimally in
physical appearance for nearly 500 million years, suggesting a very
effective chemical evolution in response to changing environmental
conditions for at least that time period. Some recognition of the
potential for utilizing biologically potent marine animal
constituents was recorded in Egypt about 2,700 BC, and by 200 BC
sea hare extracts were being used in Greece for medicinal purposes.
Such considerations, combined with the general observation that
marine organisms (especially invertebrates and sharks) rarely
develop cancer, led to the first systematic investigation of marine
animal and plant anticancer constituents.
By 1968 ample evidence had been obtained, based on the U.S.
National Cancer Institute's key experimental cancer systems, that
certain marine organisms would provide new and structurally novel
antineoplastic and/or cytotoxic agents. Analogous considerations
suggested that marine organisms could also provide effective new
drugs for other severe medical challenges, such as viral types,
that would have eluded discovery by contemporary techniques of
medicinal chemistry. Fortunately these expectations have been
realized in the intervening period. Illustrative of these successes
are discoveries of the bryostatins, dolastatins, and cephalostatins
where five members of these series of remarkable anticancer drug
candidates are either now in human clinical trial or preclinical
development.
As is well known to those presently engaged in medical research,
the time between the isolation of a new compound, and its
introduction to the market place takes at least several years in
the best case, and can take several decades, when an entity to
finance the tortuous regulatory trail is slow to appear.
Consequently, industry, in association with the government, has
devised a number of qualifying tests which serve two purposes. One
aim is to eliminate those substances whose results in the
qualifiers unequivocally demonstrate that the further expenditure
of funds thereon would be economically counterproductive. The
second, and primary aim, is to identify those substances which
demonstrate a high likelihood of success and therefore warrant the
requisite further investment necessary to obtain the data which is
required to meet the various regulatory requirements imposed by
those governments which regulate the market place into which such
substances will enter.
The present cost of obtaining such data approaches Ten Million
Dollars($10,000,000 U.S.) per compound. Economics dictate that such
an investment be made only when there is a reasonable opportunity
for it to be recovered. This opportunity can only be provided
through patent protection. Absent such protection, there will be no
such investment, and the advances in such life saving drugs will
stop.
Only two hundred years ago, many diseases ravaged humankind. Many
of these diseases have been controlled or eradicated. In the
development of the means to treat or control these diseases, work
with the appropriate common experimental animals was of critical
importance. With the various types of cancers, and with the HIV
virus, such work is presently ongoing. The research for the
treatment of various types of cancer is coordinated by the National
Cancer Institute (NCI).NCI, as a government entity, has been
charged with assisting anti-cancer research. To establish whether a
substance has anti cancer activity, NCI has established a protocol.
This protocol, which involves testing a substance against a cell
line panel containing 60 human tumor cell lines, has been verified,
and is accepted in scientific circles. This protocol, and the
established statistical means of evaluating the results obtained
therefrom have been amply described in the literature. See e.g.
Principles & Practice of Oncology PPO Updates, Volume 3, Number
10, October 1989, by Michael R. Boyd, M.D., Ph.D., for a
description of the protocol. The statistical analysis is explained
in "Display and Analysis of Patterns of Differential Activity of
Drugs Against Human Tumor Cell Lines: Development of Mean Graph and
COMPARE Algorithm" Journal of the National Cancer Institute Reports
Vol. 81, No. 14, Pg. 1088, Jul. 14, 1989, by K. D. Paull et al.
Both articles are incorporated herein by this reference as if fully
set forth.
The Constitution of the United States (Art.1, Sec.8), authorizes
Congress to establish the United States Patent and Trademark
Office(USPTO) to promote scientific advancement. This obligation
can only be fully met when the USPTO accepts current medical and
scientific realities in the area of medical research.
The Framers of the Constitution meant for the Patent system to
advance, not hamstring, scientific advancement. Cells are alive.
The impairment of human tumor cell growth is utility. The sole
right obtained by the grant of Letters Patent is that of preventing
others from exploiting the subject matter of the patent. The
recognition of cell line testing as evidence of antineoplastic
activity and hence utility can only aid research in the United
States, and will prevent the citizens of the United States from
being held hostage by foreign governments or foreign corporations,
which could otherwise procede with such projects in a less
stringent environment, especially if such research is no longer
viable in the United States.
Numerous compounds have been discovered which demonstrate
significant antineoplastic activity. As discussed above, many of
these compounds have been extracted, albeit with great difficulty,
from living creatures such as the sponge or the sea hare. However,
once the isolation and testing of such compounds has progressed, a
practical problem exists, namely, how to obtain a significant
quantity of the compound.
Unlike cinchona bark which was collected to produce quinine, and
has an excellent yield, the collection and processing of these
compounds in their natural occurring state ranges from the grossly
impractical to the utterly impossible. Even ignoring potential
ecological effects, the population of such creatures is clearly
insufficient.
Accordingly, the elucidation of the absolute structure of such an
antineoplastic compound is essential. After the absolute structure
has been determined, then means of synthesis must be discovered.
Additionally, research is essential to the determination of whether
any portion of the naturally occurring compound is irrelevant to
the desired properties thereof, which aids in determining the
simplest structure which needs to be synthesized in order to obtain
the perceived antineoplastic properties.
BRIEF DESCRIPTION OF THE INVENTION
Marine organisms, such as various species of sea hares and sponges
continue to produce numerous cyclic and linear peptides that
contain unprecedented amino acids which exhibit various important
biological activities. Such peptides comprise a promising area of
inquiry for the discovery of new anticancer drugs. Several of the
dolastatins isolated from the Indian Ocean sea hare Dolabella
auricularia have proved to be remarkably potent antineoplastic
substances representing completely new structural types. Presently,
dolastatin 10, a linear pentapeptide, has shown the most potent
antineoplastic activity profiles, of the dolastatins, against
various experimental cancer systems. Substantial research effort
has been devoted to an attempt to better understand the reasons for
this unusual efficacy. The absolute configuration of dolastatin 10
has recently been discovered. In addition, investigation as to
means of synthesis has progressed. Total synthesis has been
accomplished. Earlier, dolastatin 10 chiral isomers were prepared.
More recently these experiments were extended to the synthesis of
R-Doe-isodolastatin 10. This chiral isomer did not show any
significant difference in its human cancer cell line activity as
compared to dolastatin 10. That, in turn suggested that the
2-thiazolyl unit might not be important and could be replaced with
a simple amide.
Thus the elucidation and synthesis of selected antineoplastic
tetrapeptide phenylamides of dolastatin 10 which demonstrate
significant antineoplastic activity against a variety of human
cancer cell lines, is the object of the subject invention.
This and still further objects as shall hereinafter appear are
readily fulfilled by the present invention in a remarkably
unexpected manner as will be readily discerned from the following
detailed description of an exemplary embodiment thereof.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Marine organisms continue to produce numerous cyclic and linear
peptides that contain unprecedented amino acids which exhibit
various important biological activities. Such peptides comprise a
promising approach to discovery of new anticancer drugs. Several of
the dolastatins isolated from the Indian Ocean sea hare Dolabella
auricularia have proved to be remarkably potent antineoplastic
substances representing completely new structural types. Presently
dolastatin 10, a linear pentapeptide has shown the most potent
antineoplastic activity profiles against various experimental
cancer systems. Recently the total synthesis and the absolute
configuration of this structurally unique and biologically active
peptide was reported. This report has begun to attract increasing
interest. Accordingly research on possibly useful structural
modifications of dolastatin 10 continued.
Earlier a series of dolastatin 10 chiral isomers was prepared. More
recently these experiments were extended to synthesis of
R-Doe-isodolastatin 10. This chiral isomer did not show any
significant difference in its human cancer cell line activity as
compared to dolastatin 10. In turn that suggested that the
2-thiazolyl unit may not be too important and might be
replaced.
This synthesis of these new and potent structural modifications
took place in many steps. In each case the synthesis began with a
solution of
[2S-[2R*(.alpha.S*,.beta.S*)]]-1-[(1,1-dimethylethoxy)carbonyl]-.beta.-met
hoxy-.alpha.-methyl-2-pyrrolidinepropanoic acid (t-Boc-Dolaproine,
1, 0.144 g. 0.5 mmol), which was dissolved in 3 ml dichloromethane
distilled from CaH.sub.2. To this solution was added the respective
amine (2a-c 0.5 mmol) followed by triethylamine (0.077 ml, 0.55
mmol) and diethyl phosphorocyanidate (DEPC, 0.09 ml, 93%, 0.55
mmol, ice bath). The solution was stirred under argon for two
hours. The solvent was removed (under vacuum at room temperature)
and the residue was chromatographed (silica gel column using
hexane-acetone 3:1 as eluent). After the evaporation of solvent
from the fractions (selected by TLC) 2 ml dry dichloromethane was
added and evaporation was repeated. The residue was dried in a
desiccator under vacuum overnight to afford the amide (3a-c), which
was generally found as a viscous oil, having the structural formula
shown in FIG. 1 below.
A solution of the amide 3a-c (0.2 mmol) in dichloromethane (2 ml)
and trifluoroacetic acid (2 ml) was then stirred (ice bath under an
argon atmosphere) for two hours. The solvent was removed under
reduced pressure and the residue dissolved in toluene. Solvent was
again removed in vacuum and this operation was repeated. The
residue was dried in a desiccator (under vacuum overnight) to
afford the trifluoroacetate salt 4a-c generally found as a viscous
oil.
To a solution of the trifluoroacetate salt 4a-c (0.2 mmol) in
dichloromethane (2 ml, distilled from CaH.sub.2) was added the
tripeptide trifluoroacetate salt (5, 0.109 g, 0.2 mmol) followed by
triethylamine (0.088 ml, 0.63 mmol) and diethyl phosphorocyanidate
(DEPC, 0.036 ml, 93%, 0.22 mmol, ice bath). The solution was
stirred under argon for two hours. The solvent was removed (under
vacuum at room temperature) and the residue was chromatographed
(silica gel column using acetone-hexane 3:2 as eluent). After the
evaporation of solvent from the fractions (selected by TLC
behaviour) 2 ml of dry dichloromethane was added evaporated. The
residue was dried in a desiccator under vacuum overnight to yield a
white fluffy solid.
The following examples exemplifying the preferred embodiment of the
subject invention are offered to assist in the understanding of the
subject invention.
EXAMPLE I
Synthesis of Amides 3a-c. General Procedure A
To a solution of [2S-[2R*
(.alpha.S*,.beta.S*)]]-1-[(1,1-dimethylethoxy)carbonyl]-methoxy-.alpha.-me
thyl-2-pyrrolidinepropanoic acid (t-Boc-Dolaproine, 1, 0.144 g, 0.5
mmol) in dichloromethane (3 ml, distilled from CaH.sub.2) was added
the respective amine (2a-c 0.5 mmol) followed by triethylamine
(0.077 ml, 0.55 mmol) and diethyl phosphorocyanidate (DEPC, 0.09
ml, 93%, 0.55 mmol, ice bath) and the solution was stirred under
argon for two hours. The solvent was removed (under vacuum at room
temperature) and the residue was chromatographed (silica gel column
using hexane-acetone 3:1 as eluent).After the evaporation of
solvent from the fractions (selected by TLC) 2 ml dry
dichloromethane was added and evaporation was repeated. The residue
was dried in a desiccator under vacuum overnight to afford the
amide (3a-c), generally found as a viscous oil, having the
structural formula shown below. ##STR1##
EXAMPLE Ia
Compound 3a [2S-[2R*[1S*,
2S*]]]-2-[1-methoxy-2-methyl-3-oxo-3-benzylamino-propyl]-1-pyrrolidinecarb
oxylic acid, 1,1-dimethylethylester (3a), was synthesized from
t-Boc-Dolaproine (1) and benzylamine (2a) according to General
Procedure A as set forth in EXAMPLE I with the following
results.
Yield 3a: 0.176 g (81%) [.alpha.].sub.D.sup.25 =-42.2 (c=2.22 in
CHCl.sub.3) Anal. Calcd for C.sub.21 H.sub.32 N.sub.2 O.sub.4 M.
w.: 376.488
EXAMPLE Ib
Compound 3b [2S-[2R*[1S*,
2S*]]]-2-[1-methoxy-2-methyl-3-oxo-3-[[2-phenyl-ethyl]amino]propyl]-1-pyrr
olidinecarboxylic acid, 1,1-dimethylethylester (3b) was synthesized
from t-Boc-Dolaproine (1) and phenethylamine (2b) according to
General Procedure A set forth in EXAMPLE I, with the following
results.
Yield 3b: 0.153 g (78%) [.alpha.].sub.D.sup.25 =-37.5 (c 0.96,
CHCl.sub.3) Anal. Calcd for C.sub.22 H.sub.34 N.sub.2 O.sub.4, M.
w. :390.514
EXAMPLE Ic
Compound 3c[2S-[2R*[1S*,
2S*]]]-2-[1-methoxy-2-methyl-3-oxo-3-[[3-phenyl-propyl]amino]propyl]-1-pyr
rolidinecarboxylic acid, 1,1-dimethylethylester (3c) was
synthesized from t-Boc-Dolaproine (1) and 3-phenyl-1-propylamine
(2c) according to General Procedure A as set forth in EXAMPLE I
with the following results.
Yield 3c: 0.153 g (75%) [.alpha.].sub.D.sup.25 =-43 (c 1.8,
CHCl.sub.3) Anal. Calcd for C.sub.23 H.sub.36 N.sub.2 O.sub.4, M.
w.: 404.54
EXAMPLE II
Synthesis of Peptides 6a-c (FIGS. 2. and 3a.). General Procedure
B.
A solution of the selected amide 3a-c (0.2 mmol) in dichloromethane
(2 ml) and trifluoroacetic acid (2 ml) was stirred (ice bath under
an argon atmosphere) for two hours. The solvent was removed under
reduced pressure and the residue dissolved in toluene. Solvent was
again removed in vacuum and this operation was repeated. The
residue was dried in a desiccator (under vacuum overnight) to
afford the respective trifluoroacetate salt 4a-c as a viscous
oil.
To a solution of the selected trifluoroacetate salt 4a-c (0.2 mmol)
in dichloromethane (2 ml, distilled from CaH.sub.2) was added the
tripeptide trifluoroacetate salt (5, 0.109 g, 0.2 mmol) followed by
triethylamine (0.088 ml, 0.63 mmol) and diethyl phosphorocyanidate
(DEPC, 0.036 ml, 93%, 0.22 mmol, ice bath). The solution was
stirred under argon for two hours. The solvent was removed (under
vacuum at room temperature) and the residue was chromatographed
(silica gel column using acetone-hexane 3:2 as eluent). After the
evaporation of solvent from the fractions (selected by TLC
behaviour) 2 ml of dry dichloromethane was added evaporated. The
residue was dried in a desiccator under vacuum overnight to yield a
white fluffy solid.
FIG. 2. Trifluoroacetate salt 4a-c ##STR2##
FIG. 3a. Synthesis of Peptides 6a-c ##STR3##
EXAMPLE IIa
Compound 6a
[2S-[1[1R*(R*),2S*],2R*[1S*,2S*]]]-N,N-dimethyl-L-valyl-N-[2-methoxy-4-[2-
[1-methoxy-2-methyl-3-oxo-3-benzylamino-propyl]-1-pyrrolidinyl-1-(methylpro
pyl)-4-oxobutyl]-N-methyl-L-valineamide (6a) was synthesized from
trifluoroacetate salt 4a (from amide 3a) and tripeptide
trifluoroacetate salt 5, in accordance with General Procedure B, as
set forth in EXAMPLE II, with the following results.
Yield 6a: 112 mg (81%) M. p.: 88-93 .degree. C.
[.alpha.].sub.D.sup.25 =-40(c=0.65 in CHCl.sub.3) Anal. Calc. :
C.sub.38 H.sub.65 N.sub.5 O.sub.6 Mw.: 687.94
EXAMPLE IIb
Compound 6b
[2S-[1[1R*(R*),2S*],2R*[1S*,2S*]]]-N,N-dimethyl-L-valyl-N-[2-methoxy-4-[2-
[1-methoxy-2-methyl-3-oxo-3-[[2-phenyl-ethyl]amino]propyl]-1-pyrrolidinyl-1
-(methylpropyl)-4-oxobutyl]-N-methyl-L-valineamide (6b) was
synthesized from trifluoroacetate salt 4b (from amide 3b) and
tripeptide trifluoroacetate salt 5, in accordance with General
Procedure B, as set forth in EXAMPLE II, with the following
results.
Yield 6b: 0.115 (82%) g M. p.: 73-79.degree. C.
[.alpha.].sub.D.sup.25 =-36.9 (c 0.74, CH.sub.3 OH) Anal. Calcd for
C.sub.39 H.sub.67 N.sub.5 O.sub.6, M. w.: 701.966
EXAMPLE IIc
Compound 6c
[2S-[1[1R*(R*),2S*],2R*[1S*,2S*]]]-N,N-dimethyl-L-valyl-N-[2-methoxy-4-[2-
[1-methoxy-2-methyl-3-oxo-3-[[3-phenyl-propyl]amino]propyl]-1-pyrrolidinyl-
1-(methylpropyl)-4-oxobutyl]-N-methyl-L-valineamide (6c) was
synthesized from trifluoroacetate salt 4c (from amide 3c) and
tripeptide trifluoroacetate salt 5 in accordance with General
Procedure B, as set forth in EXAMPLE II, with the following
results.
Yield 6c: 112 mg (78%) M. p.: 106-109.degree. C.
[.alpha.].sub.D.sup.25 =-73 (c=0.1 in CHCl.sub.3) Anal. Calc.:
C.sub.40 H.sub.69 H.sub.5 O.sub.6 Mw.: 715.992
The structural modifications of dolastatin 10 whose design and
synthesis are described herein were evaluated for antineoplastic
activity. This evaluation was conducted using the standard
protocols established by National Cancer Institute (NCI) as
described above. These protocols do evaluate whether the substances
in question have antineoplastic activity against certain human
cancer cell lines derived from human cancer patients. Substantial
activity was found in those compounds tested. The antineoplastic
activity of the compounds is reported below in Table 1. The results
of the 60 cell line NCI in vitro testing of compound 6b conducted
Jun. 22, 1992, is reported below in Table 2.
TABLE 1
__________________________________________________________________________
Biological activity of Peptides 6a-c Cell type Cell line 6 a 6 b 6
c
__________________________________________________________________________
Mouse leukemia cell P-388 0.003780 0.00050 0.000440 ED-50 (mg/ml)
Ovarian OVCAR-3 <0.0001 0.000004 0.000180 CNS SF-295 <0.0001
0.000014 0.000280 Human cancer cell Renal A498 <0.0001 0.000013
0.000390 GI-50 (.mu.g/ml) Lung-NSC NCI-460 <0.0001 0.000005
0.000310 Colon KM20L2 <0.0001 0.000005 0.000330 Melanoma
SK-MEL-3 <0.0001 0.000012 0.000310 Ovarian OVCAR-3 <0.0001
0.000027 0.000540 CNS SF-295 <0.0001 0.000042 0.000790 Human
cancer cell Renal A498 <0.0001 <0.0001 <0.01 TGI
(.mu.g/ml) Lung-NSC NCI-460 <0.0001 <0.0001 <0.01 Colon
KM20L2 <0.0001 <0.0001 <0.01 Melanoma SK-MEL-3 <0.0001
<0.0001 <0.01 Ovarian OVCAR-3 <0.0001 <0.0001 <0.01
CNS SF-295 <0.0001 <0.0001 <0.01 Human cancer cells Renal
A498 <0.0001 <0.0001 <0.01 LC-50 (.mu.g/ml) Lung-NSC
NCI-460 <0.0001 <0.0001 <0.01 Colon KM20L2 <0.0001
<0.0001 <0.01 Melanoma SK-MEL-3 <0.0001 <0.0001
<0.01
__________________________________________________________________________
From the foregoing, it is readily apparent that new and useful
embodiments of the present invention have been herein described and
illustrated which fulfill all of the aforestated objectives in a
remarkably unexpected fashion. It is of course understood that such
modifications, alterations and adaptations as may readily occur to
the artisan confronted with this disclosure are intended within the
spirit of this disclosure which is limited only by the scope of the
claims appended hereto.
__________________________________________________________________________
# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii)
NUMBER OF SEQUENCES: 3 - - - - (2) INFORMATION FOR SEQ ID NO: 1: -
- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino - #acid
residues (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii)
MOLECULE TYPE: (A) DESCRIPTION: Linea - #r tetrapeptideamide - -
(iii) HYPOTHETICAL: no - - (iv) ANTI-SENSE: no - - (vi) ORIGINAL
SOURCE: synthesis - - (ix) FEATURE: (A) NAME/KEY: [2S-[1[-
#1R*(R*),2S],2R*[1S*,2S*]]]- N,N-dimethyl -
#-L-valyl-N-[2-methoxy-4-[2-[1-methoxy-2- methyl-3-oxo -
#-3-benzyl-amino-propyl]-1-pyrrolidinyl- 1-(methylpro -
#pyl)-4-oxobutyl]-N-methyl-L-valineamide (B) IDENTIFICATION METHOD:
- # by experiment using high reso - #lution nuclear magnetic
resonance and mass spectral - #techniques (C) OTHER INFORMATION: -
#this tetrapeptideamide is cell grow - #th inhibitory peptide
derivative - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1: - - Xaa
Val Xaa Xaa - - - - (2) INFORMATION FOR SEQ ID NO: 2: - - (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino - #acid residues (B)
TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: (A)
DESCRIPTION: Linea - #r tetrapeptideamide - - (iii) HYPOTHETICAL:
no - - (iv) ANTI-SENSE: no - - (vi) ORIGINAL SOURCE: synthesis - -
(ix) FEATURE: (A) NAME/KEY: [2S-[1[-
#1R*(R*),2S],2R*[1S*,2S*]]]-N,N-dimethyl L-valyl-N-[-
#2-methoxy-4-[2-[1-methoxy-2-methyl-3-oxo-3-[ [2-phenyl- -
#ethyl]amino]propyl]-1-pyrrolidinyl-1- (methylpropy -
#l)-4-oxobutyl]-N-methyl-L-valineamide (B) IDENTIFICATION METHOD: -
# by experiment using high resolution - #nuclear magnetic resonance
and mass spectral techniques (C) OTHER INFORMATION: - #this
tetrapeptideamide is cell grow - #th inhibitory peptide derivative
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2: - - Xaa Val Xaa Xaa
- - - - (2) INFORMATION FOR SEQ ID NO: 3: - - (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 4 amino - #acid residues (B) TYPE:
amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: (A)
DESCRIPTION: Linea - #r tetrapeptideamide - - (iii) HYPOTHETICAL:
no - - (iv) ANTI-SENSE: no - - (vi) ORIGINAL SOURCE: synthesis - -
(ix) FEATURE: (A) NAME/KEY: [2S-[1[- #1R*(R*),2S],2R*[1S*,2S*]]]-
N,N-dimethyl - #-L-valyl-N-[2-methoxy-4-[2-[1-methoxy-2-
methyl-3-oxo - #-3-[[3-phenyl-propyl]amino]propyl]-1- pyrrolidinyl
- #-1-(methylpropyl)-4-oxobutyl]-N-methyl- L-valineamid - #e (B)
IDENTIFICATION METHOD: - # by experiment using high reso - #lution
nuclear magnetic resonance and mass spectral - #techniques (C)
OTHER INFORMATION: - #this tetrapeptideamide is cell grow - #th
inhibitory peptide derivative - - (xi) SEQUENCE DESCRIPTION: SEQ ID
NO: - #3: - - Xaa Val Xaa Xaa
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